Culture of primary human meibomian gland cells from surgically excised eyelid tissue.

Meibomian gland dysfunction is one of the most common ocular diseases, with therapeutic treatment being primarily palliative due to our incomplete understanding of meibomian gland (MG) pathophysiology. To progress in vitro studies of human MG, this study describes a comprehensive protocol, with detailed troubleshooting, for the successful isolation, cultivation and cryopreservation of primary MG cells using biopsy-size segments of human eyelid tissue that would otherwise be discarded during surgery. MG acini were isolated and used to establish and propagate lipid-producing primary human MG cells. The primary cell viability during culture procedure was maintained through the application of Rho-associated coiled-coil containing protein kinase inhibitor (Y-27632, 10 µM) and collagen I from rat tails. Transcriptomic analysis of differentiated primary human MG cells confirmed cell origin and revealed high-level expression of many lipogenesis-related genes such as stearoyl-CoA desaturase (SCD), ELOVL Fatty Acid Elongase 1 (ELOVL1) and fatty acid synthase (FASN). Primary tarsal plate fibroblasts were also successfully isolated, cultured and cryopreserved. Established primary human MG cells and tarsal plate fibroblasts presented in this study have potential for applications in 3D models and bioengineered tissue that facilitate research in understanding of MG biology and pathophysiology.

A case of lipogranulomatous conjunctivitis in a horse.

OBJECTIVE: To describe a case of lipogranulomatous conjunctivitis in a horse. ANIMAL STUDIED: A client-owned 12-year-old Standardbred gelding presenting with chronic conjunctivitis and palpebral masses. METHODS: Complete ophthalmic examination, surgical excision, histopathology, and bacterial culture of biopsy samples were performed. RESULTS: Upper and lower eyelids of both eyes were affected, with multiple yellow-to-white nodules on the palpebral conjunctiva, adjacent to the eyelid margin. Nodule removal was achieved via partial tarsal plate excision. Histopathological examination revealed granulomatous inflammation and large droplets of presumed free lipid in the conjunctival lamina propria. The animal was diagnosed with lipogranulomatous conjunctivitis. No postoperative ocular discomfort was observed for months; however, 3 years later, new conjunctival nodules were noticed, requiring a second surgical procedure on three of the eyelids. Transient entropion in the left lower eyelid was observed 2 months after the second surgery, and no recurrence of conjunctival nodules was observed after 18 months of follow-up. CONCLUSION: To our knowledge, this is the first report of lipogranulomatous conjunctivitis in horses.

Morphology and volume of Meibomian glands ex vivo pre and post partial tarsal plate excision, cryotherapy and laser therapy in the dog using microCT.

OBJECTIVE: To determine the morphology and volume of Meibomian glands (MG) of dogs with microCT before and after partial tarsal plate excision (PTPE), cryotherapy, and laser therapy. PROCEDURE: MicroCT scans were made of 12 upper lids (ULs) and lower lids (LLs) of 12 dogs. After undergoing PTPE, 10 ULs and LLs were scanned again, and one UL and one LL was scanned after laser therapy and one UL and one LL after cryotherapy. RESULTS: The length of the area containing MGs did not change pre- and post-PTPE, and cryo- or laser therapy. The mean number of MGs in the ULs and LLs was 30.50 and 29.42, respectively, and did not change during the procedures. The average length of one individual MG was 2.60 mm. The mean volume of MGs in the 10 ULs and LLs pre-PTPE was 21.45 and 17.2 mm3 , respectively, and 12.84 and 11.25 mm3 in the UL and LL after PTPE, respectively. The mean volume of MGs decreased from 29.78 mm3 precryotherapy to 28.91 mm3 post-treatment and in the lower eyelid from 22.87 to 22.4 mm3 after cryotherapy. The mean volume of MGs in the UL and LL before laser therapy was 8.95 and 6.78 mm3 , respectively, and after 9.25 and 6.38 mm3 , respectively. CONCLUSION: MicroCT is a valuable tool to determine the morphology and the volume of MGs and to demonstrate changes that occur after PTPE, laser-, and cryotherapy. There is no need for additional preparation, such as staining, of the specimen prior to scanning.

Lifting effect of onabotulinumtoxinA in patients treated for glabellar and crow's feet rhytids.

OnabotulinumtoxinA injection can enhance the appearance of the eyes. This study evaluated the lifting effect of onabotulinumtoxinA treatment of the eye area. A retrospective, single-center audit of patients treated for glabellar and crow's feet rhytids was undertaken. Standardized photographs taken before and 2-4 weeks after treatment were assessed. Computer-based measurements were made of the height of the visible superior tarsal plate (STP) and brow (measured from upper eyelid lash edge to eyebrow base) at rest and maximum frown. One hundred patients were included (96 females; mean age: 46.2 ± 8.8 years). Mean onabotulinumtoxinA doses were 14.95 ± 0.61 units (glabella) and 17.83 ± 2.74 units (crow's feet). There were significant post-treatment increases in mean STP height at rest (left side, 0.76 ± 0.99 mm, p = .02; right side, 0.79 ± 0.94 mm, p = .01) and maximum frown (left, 2.25 ± 1.52 mm, p < .0001; right, 1.87 ± 1.34 mm, p < .0001), and mean brow height at rest (left side, 1.54 ± 1.49 mm, p = .0006; right side, 1.47 ± 1.84 mm, p = .0009) and maximum frown (left, 4.37 ± 2.29 mm, p < .0001; right, 4.16 ± 1.88 mm, p < .0001). OnabotulinumtoxinA injection is effective for elevating brow position and increasing STP show.

Evaluation of partial tarsal plate excision using a transconjunctival approach for the treatment of distichiasis in dogs.

PURPOSE: To evaluate the clinical outcome, possible complications, and recurrence rate of distichiasis in dogs treated with partial tarsal plate excision (PTPE) technique using a transconjunctival approach. METHODS: Retrospective study including 17 client-owned canine patients affected with distichiasis and presenting with associated clinical signs (ie, blepharospasm, epiphora, chronic keratoconjunctivitis, or corneal ulceration) that underwent surgical removal of the aberrant lashes using a PTPE technique between January 2018 and February 2019. Data collected included breed, age, sex, affected eyelid(s), number of distichia, and tear film breakup time (TBUT). Resected cilia-bearing tarsoconjunctival strips were submitted for histopathological analysis. RESULTS: Thirty eyes (52 eyelids) from 17 dogs were included in the study. The median age was 688 days (range 118-4243 days). A successful outcome, defined as complete resolution of clinical signs attributable to the distichia, occurred in all eyes after a single procedure, with a mean follow-up time of 239 days (range 69-480 days). Appearance of new distichia occurred in 14/30 eyes (46.3%), and of these, three eyes needed a new PTPE procedure. Recurrence of the distichia only occurred in one eye (3.3%) which was asymptomatic. Following surgery, TBUT decreased below the normal value in 7/24 eyes (29.1%) although none developed clinical signs of qualitative tear film deficiency. Post-operative complications included trichiasis and cicatricial entropion, which developed in two eyes (6.6%), and these were successfully managed with corrective eyelid surgery. CONCLUSIONS: Partial tarsal plate excision, using a transconjunctival approach, had an excellent clinical outcome with a low incidence of complications.

[Experimental justification of the use of a polymeric implant in reconstructive and regenerative surgery of the sclera and tarsal plate]. / Éksperimental'noe obosnovanie ispol'zovaniia polimernogo implantata v rekonstruktivno-vosstanovitel'noi khirurgii sklery i tarzal'noi plastinki.

INTRODUCTION: Congenital and acquired diseases, as well as traumatic injuries of the sclera and tarsal plate can lead to serious complications up to the complete loss of visual functions and loss of the eye as an organ if not treated timely. Due to that, it becomes necessary to use biological and synthetic materials during reconstructive and regenerative surgeries to replace the defects and reinforce the carcass properties of the sclera and tarsal plate. PURPOSE: To compare experimental and morphological features of the response of the eye and surrounding tissues to implantation of the synthetic polymeric implant for reconstructive and regenerative surgery (PERRS) 'Reperen-6' and biological xenopericardium implant taken from cattle on the sclera in animal experiment. MATERIAL AND METHODS: The studies were conducted on 12 chinchilla rabbits. PERRS 'Reperen-6' was implanted on the surface of the sclera (into the subtenon space) of left eyes of 6 rabbits from the main group. Similar surgeries with biological xenopericardium implant taken from cattle were performed on the left eyes of 6 rabbits from the comparison group. The right eyes of each animal from both groups remained intact. After the surgery, we performed examination of the animals and acquisition of the material (the eyes and surrounding tissue) in 16, 34 and 68 days. Enucleated eyes were fixed in 10% buffered formalin solution with subsequent preparation of histologic specimen according to standard procedure. CONCLUSION: The experimental and morphological studies showed that the postoperative period went smoother in the main group of animals. Thanks to its structure, PERRS 'Reperen-6' fixed tightly to the sclera and surrounding structures forming a firm 'own tissue-implant' complex. The polymeric implant 'Reperen-6' can be recommended for reconstructive and regenerative surgeries to reinforce the connective tissue structures in pathological conditions of sclera and appendages of the eyes.

A new perspective in oculoplastic surgical management of symptomatic distichiasis in lymphedema-distichiasis syndrome.

Lymphedema-distichiasis syndrome (LDS) is an autosomal dominant condition associated with FOXC2 mutations. Patients with distichiasis are mostly symptomatic, and efforts to deal with their ocular complaints comprise of electrolysis, cryotherapy and a variety of surgical techniques. We describe an enhanced surgical technique for a case of symptomatic distichiasis of the right eye with scarred, irregular eyelid margins secondary to initial cryotherapy, whereby the distorted tarsus was excised to remove the aberrant hair follicles, the levator palpebrae superioris was released to extend the upper lid and prevent lagophthalmos and a mucous membrane graft was used to cover the exposed portion of the tarsal plate. At 14 months follow up, the lid cosmesis and position remained satisfactory, with no infection or rejection of the mucous membrane graft. Therefore, this surgical technique provides a sound option for symptomatic distichiasis, where cryotherapy can cause lid irregularity and keratinization.

Anatomical description of the upper tarsal plate for reconstruction.

BACKGROUND: Reconstruction of the upper eyelids following traumatic, congenital and tumor surgeries is often difficult owing to a variety of reasons including the influence of the lacrimal system, visual system and aesthetic appearance. In most cases of the reconstruction in the upper eyelid tarsal plate is the main anatomical area that should be protected against the damage. The aim of this study is to investigate the types and the measurements of the tarsal plate of the upper eyelids in Anatolian population. METHODS: Fifty cadaver upper eyelids, tarsal plates were exposed to investigate the location, shape, position and their relationships to the upper eyelid. Their morphometric details such as linear analyses (vertical and horizontal) and ratio analyses were studied. As for the shape of the upper tarsal plate, it was categorized into three distinct types: sickle, trapezoid, and triangular type. RESULTS: The vertical height of the upper tarsal plate was the greatest at the central point 10.6 ± 1.1 mm, followed by the lateral point (7.81 ± 1.0 mm), and medial point (6.2 ± 0.8 mm) medially. The mean medial width of the upper tarsal plate was measured as 37.6 ± 4.1 mm and the lower width as 38.5 ± 4.6 mm. The base-central height ratio of the upper tarsal plate was approximately 0.28. For the upper eyelids, the shapes of tarsal plates were observed as sickle (48%), trapezoid (28%) and triangular (24%). Sickle type was the most frequent upper eyelid type. CONCLUSIONS: Considering the shielding function of the upper eyelid, tarsal plate, serves as fibrocartilaginous skeleton of the upper tarsal plate. In successful lid oculoplastic reconstructive surgery, tarsal plate may be restored by evaluating each patient individually to have symmetrical and youthful eyes. Although sickle tarsal plate is the most frequent type, personalized treatment requires measurement and classification. The findings manifest the necessity of oculoplastic surgical treatment peculiar to each individual. LEVEL OF EVIDENCE: I, Randomized controlled trial.

Perturbed meibomian gland and tarsal plate morphogenesis by excess TGFα in eyelid stroma.

Transforming growth factor alpha (TGFα) belongs to the epidermal growth factor (EGF) family and is known to play an important role during eyelid morphogenesis. In this study, we showed that ectopic expression of TGFα in the stroma of Kera-rtTA/tet-O-TGFα bitransgenic mice results in precocious eye opening, abnormal morphogenesis of the meibomian gland, tendon and tarsal plate malformation and epithelium hyperplasia. TGFα did not change proliferation and differentiation of meibocytes, but promoted proliferation and inhibited differentiation of the tarsal plate tenocytes. These results suggest that proper formation of the tendon and tarsal plate in the mouse eyelid is required for normal morphogenesis of the meibomian gland.

Reconstruction of large upper eyelid defects with a free tarsal plate graft and a myocutaneous pedicle flap plus a free skin graft.

PURPOSE: To review and present the results of a one-step method employing a free tarsal plate graft and a myocutaneous pedicle flap plus a free skin graft for reconstruction of large upper eyelid defects after tumour surgery. METHODS: This was a retrospective case-series of 8 patients who underwent reconstruction of the upper eyelid after tumour removal. The horizontal defect involved 50-75% of the lid (3 pts.), more than 75% (3 pts.), and more than 75% plus the lateral canthus (2 pts.). The posterior lamella was reconstructed with contralateral upper eyelid tarsal plate. The anterior lamella was reconstructed with a laterally based myocutaneous pedicle flap in 7 patients, leaving a raw surface under the brow which was covered with a free skin graft. In 1 patient with little skin left under the brow, the anterior lamella was reconstructed with a bi-pedicle orbicularis muscle flap together with a free skin graft. RESULTS: All patients healed without necrosis, did not suffer from lagophthalmos, achieved reasonable cosmesis, and did not need lubricants. In one patient, a contact lens was necessary for three weeks because of corneal erosion. One patient still needs a contact lens 3 months after excision to avoid eye discomfort. CONCLUSION: Large upper eyelid defects can be reconstructed with a free tarsal plate graft and a laterally based myocutaneous pedicle flap in combination with a free skin graft. Two-step procedures can probably be avoided in most cases.

Biomaterials and tissue engineering strategies for posterior lamellar eyelid reconstruction: Replacement or regeneration?

Reconstruction of posterior lamellar eyelids remains challenging due to their delicate structure, highly specialized function, and cosmetic concerns. Current clinically available techniques for posterior lamellar reconstruction mainly focus on reconstructing the contour of the eyelids. However, the posterior lamella not only provides structural support for the eyelid but also offers a smooth mucosal surface to facilitate globe movement and secrete lipids to maintain ocular surface homeostasis. Bioengineered posterior lamellar substitutes developed via acellular or cellular approaches have shown promise as alternatives to current therapies and encouraging outcomes in animal studies and clinical conditions. Here, we provide a brief reference on the current application of autografts, biomaterials, and tissue-engineered substitutes for posterior lamellar eyelid reconstruction. We also shed light on future challenges and directions for eyelid regeneration strategies and offer perspectives on transitioning replacement strategies to regeneration strategies for eyelid reconstruction in the future.

Azithromycin-carrying and microtubule-orientated biomimetic poly (lactic-co-glycolic acid) scaffolds for eyelid reconstruction.

Introduction: Tarsal plate repair is the major challenge of eyelid reconstruction for the oculoplastic surgeon. The ideal synthetic tarsal plate substitute should imitate the microstructure and mechanical strength of the natural eyelid. The aim of this work was to develop a novel bionic substitute for eyelid reconstruction. Methods: Three types of poly(lactic-co-glycolic acid) (PLGA) scaffolds (random, oriented, and azithromycin-loaded oriented scaffolds) were prepared using an improved thermal-induced phase separation technique. The microstructure of the scaffolds was examined by scanning electron microscopy. In vitro cytotoxicity was assessed using scaffold extracts. Fibroblast and primary rat meibomian gland epithelial cells (rMGCs) were cultured within the scaffolds, and their behavior was observed using fluorescence staining. Three types of PLGA scaffolds were implanted into rabbit eyelid defect in vivo to evaluate their inductive tissue repair function. Results: We successfully fabricated three types of PLGA scaffolds with varying pore architectures, and the axially aligned scaffold demonstrated interconnected and vertically parallel channels. In vitro cytotoxicity tests using scaffold extracts revealed no apparent cytotoxicity. Fluorescence staining showed that both Fibroblast and rMGCs could adhere well onto the pore walls, with fibroblast elongating along the axially aligned porous structure. At 8 weeks post-implantation, all scaffolds were well integrated by fibrovascular tissue. The axially aligned scaffold groups exhibited faster degradation compared to the random scaffold group, with smaller fragments surrounded by mature collagen fibers. Conclusion: The study found that the axially aligned scaffolds could well support and guide cellular activities in vitro and in vivo. Moreover, the axially aligned scaffold group showed a faster degradation rate with a matched integration rate compared to the random scaffold group. The findings suggest that the oriented scaffold is a promising alternative for eyelid tarsal plate substitutes.

Ptosis Correction: Our Modification and Experience.

Background Congenital ptosis not only results in an asymmetric facial appearance but can lead to permanent visual disturbances if not addressed at an appropriate time. Crawford used fascia lata for suspension of the eyelid to frontalis muscle, which remains a standard procedure for congenital ptosis correction to date, with an acceptable recurrence rate due to graft slippage. There are many modifications in this technique to reduce this complication; hence, in this study, we share our experience of a modification to improve the outcomes. Methodology This retrospective study was conducted at a private tertiary care hospital in Karachi for 10 years. In total, 26 patients fulfilled our inclusion criteria. All patients underwent a modified Crawford's procedure under general anesthesia. Results In this study, the male-to-female ratio was 1:1.5. In total, 17 (65%) patients had unilateral ptosis. The mean age of presentation was 7 ± 3 years. All of our patients had poor levator function (<5 mm excursion) with a mean of 3 mm and mean grade of ptosis of 4 ± 1.6 mm. The mean preoperative marginal reflex distance (MRD) was +1.8 ± 0.6 mm. In this study, the patients had a mean postoperative MRD of 4.2 ± 0.7 mm at the four-week follow-up. Conclusions Although Crawford's procedure gives promising results for ptosis correction, suturing the fascial sling to the tarsal plate ensures good anchorage and prevents relapse.

Conjunctival Autograft for Bilateral Tarsal Keratinization in a Case of Chronic Vernal Keratoconjunctivitis.

This report describes the clinical features and management in a case of vernal keratoconjunctivitis (VKC) with bilateral tarsal conjunctival keratinization. A 32-year-old male presented with VKC since childhood that had exacerbated in the eight years prior to presentation. Examination revealed partial limbal stem cell deficiency in both eyes, with keratinization of the superior tarsal conjunctiva. The corresponding areas of the cornea exhibited punctate keratopathy in both eyes. To address this, the patient underwent excision of the conjunctival keratinization in both eyes. The resultant bare areas were covered with conjunctival autografts (CAGs). Postoperatively, the grafts were well apposed, and there was no recurrence of keratinization observed during the period of follow-up of four years. Resolution of corneal epitheliopathy was also noted. Although keratinization can occur in eyes with VKC, it is usually limited to the bulbar conjunctival areas. This is the first report of tarsal conjunctival keratinization in such cases. Milder cases may be observed or managed with scleral contact lenses. In more severe forms, there is associated corneal epitheliopathy, which may progress to corneal vascularization and scarring. Surgical excision of the lesion is recommended in these eyes. Following excision, several options exist to cover the bare area, which include a CAG, an amniotic membrane, or an oral mucous membrane. Of these, a CAG is an autologous tissue that can be harvested with a simple surgical technique and yields stable long-term results. Thus, tarsal conjunctival keratinization is a rare complication of chronic VKC. Excision of the lesion followed by a CAG is a viable approach for treatment, which reestablishes and maintains a stable ocular surface.

Efficient DNA/RNA extraction from tarsal plates by SK mill, a freeze-crush apparatus.

The tarsal plate is an eyelid tissue that maintains lid structure from inside the upper/lower eyelids, and it surrounds the meibomian glands and supports their unique secretion mechanism. Sebaceous carcinoma, a malignant eyelid tumour, can sometimes develop from the meibomian glands and is usually excised together with the tarsal plate during surgery, so the tarsal plate serves as a control research tissue. However, since the plate is thick, hard and heterogeneous with few cells, obtaining enough genomic DNA and/or total RNA is often difficult. Therefore, we attempted to establish an efficient protocol to obtain DNA and RNA simultaneously by comparing the combinations of homogenization (mortar/pestle, pellet pestle or SK mill) and purification (organic solvent or spin column) methods using rabbit tarsal plates. Based on the yield, quality and hands-on time, the SK mill and spin column was found to be the most efficient combination. We then applied the established protocol to extract DNA/RNA from six human tarsal-plate samples and succeeded in generating high-quality exome and transcriptome datasets via a next-generation sequencer with sufficient coverage and meibomian gland-specific expression of representative genes, respectively. Our new findings will provide ideal reference data for future genetic and gene-expression studies of sebaceous carcinoma.

Total or subtotal replacement of tarsal plate by novel silicone plate for upper eyelid reconstruction in malignant tumors.

PURPOSE: : To evaluate the cost, safety, surgical outcome, and efficacy of modified Cutler-Beard eyelid reconstruction utilizing a novel silicone plate as a tarsal plate replacement in the repair of 60% to 100% eyelid defects following the excision of large malignant tumors. METHODS: : A prospective, noncomparative, interventional study of 30 eyes was done over 3 years. Fourteen patients were female, and 16 patients were male. In all the cases, a silicone plate, the synthetic, artificial tarsal plate, was utilized for a total or subtotal replacement of the tarsal plate. The created defect was measured in mm (length and width) and later expressed in percentage. Pre- and postoperative action of levator palpebrae superioris (LPS) was measured. Pre- and postoperative measurements of the margin-to-margin reflex distance (MRD1) were noted. RESULTS: : Preoperative LPS action was 1.23 ± 1.35 mm, whereas postoperative LPS actions at the end of 1 week and 18 months were 11. 8 ± 0.88 mm and 13.53 ± 0. 73 mm, respectively. Preoperative MRD1 was - 3.0 ± 1.144 mm, whereas postoperative MRD1 values at the end of 1 week and 18 months were 2.18 ± 0.27 mm and 4.16 mm ± 0.35, respectively. The mean created defect after the removal of the tumor was 87.3% ±11.10. The mean length of the silicone plate implanted in this study was 27.53 ± 2.48 mm. The follow-up period for the study participants was 18 months. CONCLUSION: : The synthetic novel silicone plate was successful as a tarsal plate replacement. A second surgical site for ear cartilage harvesting is avoided. Cadaver transfer of Achilles tendon carries the risk of transmission of communicable diseases, for example, hepatitis B and HIV. Silicone is an inert, nonreacting, and tissue-tested material, thus eliminating the possibility of graft rejection. This material is readily available and cost-effective. The novel silicone plate is considered to be the most promising alternative material as a tarsal replacement in the future generation.

Corrigendum: 3D-Printed Poly-Caprolactone Scaffolds Modified With Biomimetic Extracellular Matrices for Tarsal Plate Tissue Engineering.

[This corrects the article DOI: 10.3389/fbioe.2020.00219.].

3D-Printed Poly-Caprolactone Scaffolds Modified With Biomimetic Extracellular Matrices for Tarsal Plate Tissue Engineering.

Tarsal plate regeneration has always been a challenge in the treatment of eyelid defects. The commonly used clinical treatments such as hard palate mucosa grafts cannot achieve satisfactory repair effects. Tissue engineering has been considered as a promising technology. However, tarsal plate tissue engineering is difficult to achieve due to its complex structure and lipid secretion function. Three-dimensional (3D) printing technology has played a revolutionary role in tissue engineering because it can fabricate complex scaffolds through computer aided design (CAD). In this study, it was novel in applying 3D printing technology to the fabrication of tarsal plate scaffolds using poly-caprolactone (PCL). The decellularized matrix of adipose-derived mesenchymal stromal cells (DMA) was coated on the surface of the scaffold, and its biofunction was further studied. Immortalized human SZ95 sebocytes were seeded on the scaffolds so that neutral lipids were secreted for replacing meibocytes. In vitro experiments revealed excellent biocompatibility of DMA-PCL scaffolds with sebocytes. In vivo experiments revealed excellent sebocytes proliferation on the DMA-PCL scaffolds. Meanwhile, sebocytes seeded on the scaffolds secreted abundant neutral lipid in vitro and in vivo. In conclusion, a 3D-printed PCL scaffold modified with DMA was found to be a promising substitute for tarsal plate tissue engineering.

Regeneration of different types of tissues depends on the interplay of stem cells-laden constructs and microenvironments in vivo.

The ability of repair and regeneration of tissues or organs has been significantly improved by using biomaterials-based constructs. Our previous studies found the regeneration of both articular cartilage and subchondral bone by implantation of a poly(lactide-co-glycolide) (PLGA)/fibrin gel/bone marrow stem cells (BMSCs)/(lipofectamine/pDNA-transforming growth factor (TGF)-ß1) construct in vivo, without the step of pre-induced differentiation of the laden stem cells in vitro. To substantiate the ability to regenerate multi-types of tissues by the same constructs, in this study the constructs were implanted into three types of tissues or tissue defects in vivo, including subcutaneous fascia layer, and ear cartilage and eyelid tarsal plate defects. The ear cartilage and eyelid tarsal plate defects were fully regenerated 8 w post-implantation, showing a similar morphology to the corresponding native tissues. In the neo ear cartilage, abundant chondrocytes with obvious lacunas and cartilage-specific extracellular matrices (ECMs) were found. Neo eyelid tarsal plate with mature meibomian gland acinar units was regenerated. Furthermore, expressions of the ECMs-specific genes and proteins, as well as the cell behavior modulatory factors, Sry related HMG box 9 (Sox9) and TGF-ß1 were significantly up-regulated in the regenerated ear cartilages and eyelid tarsal plate than those in the subcutaneously implanted constructs, which were filled with fibrocytes, inflammatory cells, obvious vascularization and slight ECMs deposition. These results confirm firmly the ability to regenerate multi-types of tissues by a stem cells-laden construct via adapting to the microenvironments of corresponding tissues.

Best technique for upper lid eversion.

AIM: Lid eversion is an essential component of contact lens aftercare. Hence, this study determined the best method of lid eversion based on three criteria: comfort, speed of administration and the area of the palpebral conjunctiva exposed. METHOD: Twenty-five participants (aged 20-34) had 6 different techniques applied in random order by the same clinician on two separate occasions: three involving a cotton bud placed on the extended upper eyelid either centrally, at the top of the tarsal plate or off-centre; one using the wooden end of the bud placed at the top of the tarsal plate; one using the clinician's index finger to evert the lid; and one using a silicone rubber, finger-shaped substitute. The participants judged the degree of discomfort of each technique on a visual analogue scale. The time to complete the task was timed with a stop-watch and the area of exposed palpebral conjunctiva was captured with a digital slit lamp and assessed using image analysis. RESULTS: There was no difference between the initial lid eversion or subsequent repeat in terms of comfort (F = 0.304, p = 0.586), time to complete (F = 3.075, p = 0.092) or area exposed (F = 2.311, p = 0.142). Lid eversion using fingers alone or the silicone substitute everter were similar in comfort (p = 0.312), being the most comfortable methods, with off-centre cotton bud eversion or the wooden end of the cotton bud the least comfortable techniques (F = 17.480, p < 0.001). The quickest method to perform was the wooden end of the cotton bud, followed by the silicone everter (F = 17.522, p < 0.001). The area of exposed palpebral conjunctiva was greatest using the silicone everter (F = 28.199, p < 0.001). CONCLUSIONS: Lid eversion had repeatable results, with the silicone everter placed at the top of the tarsal plate the most comfortable for the patient, quick to perform and exposed a greater area of tarsal plate than other techniques and therefore is recommended to clinicians.